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. 2002 May;82(5):2635–2644. doi: 10.1016/S0006-3495(02)75605-1

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin.

Filip Meersman 1, László Smeller 1, Karel Heremans 1
PMCID: PMC1302052  PMID: 11964250

Abstract

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).

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Selected References

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  1. Bondos S. E., Sligar S., Jonas J. High-pressure denaturation of apomyoglobin. Biochim Biophys Acta. 2000 Jul 14;1480(1-2):353–364. doi: 10.1016/s0167-4838(00)00088-1. [DOI] [PubMed] [Google Scholar]
  2. Booth D. R., Sunde M., Bellotti V., Robinson C. V., Hutchinson W. L., Fraser P. E., Hawkins P. N., Dobson C. M., Radford S. E., Blake C. C. Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature. 1997 Feb 27;385(6619):787–793. doi: 10.1038/385787a0. [DOI] [PubMed] [Google Scholar]
  3. Byler D. M., Susi H. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers. 1986 Mar;25(3):469–487. doi: 10.1002/bip.360250307. [DOI] [PubMed] [Google Scholar]
  4. Carpenter J. F., Kendrick B. S., Chang B. S., Manning M. C., Randolph T. W. Inhibition of stress-induced aggregation of protein therapeutics. Methods Enzymol. 1999;309:236–255. doi: 10.1016/s0076-6879(99)09018-7. [DOI] [PubMed] [Google Scholar]
  5. Carrell R. W., Gooptu B. Conformational changes and disease--serpins, prions and Alzheimer's. Curr Opin Struct Biol. 1998 Dec;8(6):799–809. doi: 10.1016/s0959-440x(98)80101-2. [DOI] [PubMed] [Google Scholar]
  6. Damaschun G., Damaschun H., Fabian H., Gast K., Kröber R., Wieske M., Zirwer D. Conversion of yeast phosphoglycerate kinase into amyloid-like structure. Proteins. 2000 May 15;39(3):204–211. doi: 10.1002/(sici)1097-0134(20000515)39:3<204::aid-prot20>3.0.co;2-8. [DOI] [PubMed] [Google Scholar]
  7. Dong A., Randolph T. W., Carpenter J. F. Entrapping intermediates of thermal aggregation in alpha-helical proteins with low concentration of guanidine hydrochloride. J Biol Chem. 2000 Sep 8;275(36):27689–27693. doi: 10.1074/jbc.M005374200. [DOI] [PubMed] [Google Scholar]
  8. Englander S. W. Protein folding intermediates and pathways studied by hydrogen exchange. Annu Rev Biophys Biomol Struct. 2000;29:213–238. doi: 10.1146/annurev.biophys.29.1.213. [DOI] [PubMed] [Google Scholar]
  9. Englander S. W., Sosnick T. R., Englander J. J., Mayne L. Mechanisms and uses of hydrogen exchange. Curr Opin Struct Biol. 1996 Feb;6(1):18–23. doi: 10.1016/s0959-440x(96)80090-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Evans S. V., Brayer G. D. High-resolution study of the three-dimensional structure of horse heart metmyoglobin. J Mol Biol. 1990 Jun 20;213(4):885–897. doi: 10.1016/S0022-2836(05)80270-0. [DOI] [PubMed] [Google Scholar]
  11. Fabian H., Mantsch H. H. Ribonuclease A revisited: infrared spectroscopic evidence for lack of native-like secondary structures in the thermally denatured state. Biochemistry. 1995 Oct 17;34(41):13651–13655. doi: 10.1021/bi00041a046. [DOI] [PubMed] [Google Scholar]
  12. Fabian H., Schultz C., Naumann D., Landt O., Hahn U., Saenger W. Secondary structure and temperature-induced unfolding and refolding of ribonuclease T1 in aqueous solution. A Fourier transform infrared spectroscopic study. J Mol Biol. 1993 Aug 5;232(3):967–981. doi: 10.1006/jmbi.1993.1442. [DOI] [PubMed] [Google Scholar]
  13. Ferrão-Gonzales A. D., Souto S. O., Silva J. L., Foguel D. The preaggregated state of an amyloidogenic protein: hydrostatic pressure converts native transthyretin into the amyloidogenic state. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6445–6450. doi: 10.1073/pnas.97.12.6445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Floriano W. B., Nascimento M. A., Domont G. B., Goddard W. A., 3rd Effects of pressure on the structure of metmyoglobin: molecular dynamics predictions for pressure unfolding through a molten globule intermediate. Protein Sci. 1998 Nov;7(11):2301–2313. doi: 10.1002/pro.5560071107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fändrich M., Fletcher M. A., Dobson C. M. Amyloid fibrils from muscle myoglobin. Nature. 2001 Mar 8;410(6825):165–166. doi: 10.1038/35065514. [DOI] [PubMed] [Google Scholar]
  16. Gilmanshin R., Williams S., Callender R. H., Woodruff W. H., Dyer R. B. Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3709–3713. doi: 10.1073/pnas.94.8.3709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goossens K., Smeller L., Frank J., Heremans K. Pressure-tuning the conformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy. Eur J Biochem. 1996 Feb 15;236(1):254–262. doi: 10.1111/j.1432-1033.1996.00254.x. [DOI] [PubMed] [Google Scholar]
  18. Griko YuV, Kutyshenko V. P. Differences in the processes of beta-lactoglobulin cold and heat denaturations. Biophys J. 1994 Jul;67(1):356–363. doi: 10.1016/S0006-3495(94)80488-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haris P. I., Chapman D. The conformational analysis of peptides using Fourier transform IR spectroscopy. Biopolymers. 1995;37(4):251–263. doi: 10.1002/bip.360370404. [DOI] [PubMed] [Google Scholar]
  20. Hawley S. A. Reversible pressure--temperature denaturation of chymotrypsinogen. Biochemistry. 1971 Jun 22;10(13):2436–2442. doi: 10.1021/bi00789a002. [DOI] [PubMed] [Google Scholar]
  21. Heremans K., Smeller L. Protein structure and dynamics at high pressure. Biochim Biophys Acta. 1998 Aug 18;1386(2):353–370. doi: 10.1016/s0167-4838(98)00102-2. [DOI] [PubMed] [Google Scholar]
  22. Holzbaur I. E., English A. M., Ismail A. A. FTIR study of the thermal denaturation of horseradish and cytochrome c peroxidases in D2O. Biochemistry. 1996 Apr 30;35(17):5488–5494. doi: 10.1021/bi952233m. [DOI] [PubMed] [Google Scholar]
  23. Huang G. S., Oas T. G. Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent. Biochemistry. 1996 May 21;35(20):6173–6180. doi: 10.1021/bi960250l. [DOI] [PubMed] [Google Scholar]
  24. Hummer G., Garde S., García A. E., Paulaitis M. E., Pratt L. R. The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1552–1555. doi: 10.1073/pnas.95.4.1552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ismail A. A., Mantsch H. H., Wong P. T. Aggregation of chymotrypsinogen: portrait by infrared spectroscopy. Biochim Biophys Acta. 1992 May 22;1121(1-2):183–188. doi: 10.1016/0167-4838(92)90353-f. [DOI] [PubMed] [Google Scholar]
  26. Jackson M., Mantsch H. H. The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol. 1995;30(2):95–120. doi: 10.3109/10409239509085140. [DOI] [PubMed] [Google Scholar]
  27. Jennings P. A., Wright P. E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science. 1993 Nov 5;262(5135):892–896. doi: 10.1126/science.8235610. [DOI] [PubMed] [Google Scholar]
  28. Jonas J., Ballard L., Nash D. High-resolution, high-pressure NMR studies of proteins. Biophys J. 1998 Jul;75(1):445–452. doi: 10.1016/S0006-3495(98)77532-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kaposi A. D., Fidy J., Manas E. S., Vanderkooi J. M., Wright W. W. Horseradish peroxidase monitored by infrared spectroscopy: effect of temperature, substrate and calcium. Biochim Biophys Acta. 1999 Nov 16;1435(1-2):41–50. doi: 10.1016/s0167-4838(99)00206-x. [DOI] [PubMed] [Google Scholar]
  30. Kim Y., Wall J. S., Meyer J., Murphy C., Randolph T. W., Manning M. C., Solomon A., Carpenter J. F. Thermodynamic modulation of light chain amyloid fibril formation. J Biol Chem. 2000 Jan 21;275(3):1570–1574. doi: 10.1074/jbc.275.3.1570. [DOI] [PubMed] [Google Scholar]
  31. Krimm S., Bandekar J. Vibrational analysis of peptides, polypeptides, and proteins. V. Normal vibrations of beta-turns. Biopolymers. 1980 Jan;19(1):1–29. doi: 10.1002/bip.1980.360190102. [DOI] [PubMed] [Google Scholar]
  32. Martínez A., Haavik J., Flatmark T., Arrondo J. L., Muga A. Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect of iron/catecholamine binding and phosphorylation. J Biol Chem. 1996 Aug 16;271(33):19737–19742. doi: 10.1074/jbc.271.33.19737. [DOI] [PubMed] [Google Scholar]
  33. Mombelli E., Afshar M., Fusi P., Mariani M., Tortora P., Connelly J. P., Lange R. The role of phenylalanine 31 in maintaining the conformational stability of ribonuclease P2 from Sulfolobus solfataricus under extreme conditions of temperature and pressure. Biochemistry. 1997 Jul 22;36(29):8733–8742. doi: 10.1021/bi970467v. [DOI] [PubMed] [Google Scholar]
  34. Nash D. P., Jonas J. Structure of the pressure-assisted cold denatured state of ubiquitin. Biochem Biophys Res Commun. 1997 Sep 18;238(2):289–291. doi: 10.1006/bbrc.1997.7308. [DOI] [PubMed] [Google Scholar]
  35. Oliveira A. C., Ishimaru D., Gonçalves R. B., Smith T. J., Mason P., Sá-Carvalho D., Silva J. L. Low temperature and pressure stability of picornaviruses: implications for virus uncoating. Biophys J. 1999 Mar;76(3):1270–1279. doi: 10.1016/S0006-3495(99)77290-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Panick G., Malessa R., Winter R., Rapp G., Frye K. J., Royer C. A. Structural characterization of the pressure-denatured state and unfolding/refolding kinetics of staphylococcal nuclease by synchrotron small-angle X-ray scattering and Fourier-transform infrared spectroscopy. J Mol Biol. 1998 Jan 16;275(2):389–402. doi: 10.1006/jmbi.1997.1454. [DOI] [PubMed] [Google Scholar]
  37. Panick G., Vidugiris G. J., Malessa R., Rapp G., Winter R., Royer C. A. Exploring the temperature-pressure phase diagram of staphylococcal nuclease. Biochemistry. 1999 Mar 30;38(13):4157–4164. doi: 10.1021/bi982608e. [DOI] [PubMed] [Google Scholar]
  38. Panick G., Winter R. Pressure-induced unfolding/refolding of ribonuclease A: static and kinetic Fourier transform infrared spectroscopy study. Biochemistry. 2000 Feb 22;39(7):1862–1869. doi: 10.1021/bi992176n. [DOI] [PubMed] [Google Scholar]
  39. Privalov P. L. Cold denaturation of proteins. Crit Rev Biochem Mol Biol. 1990;25(4):281–305. doi: 10.3109/10409239009090612. [DOI] [PubMed] [Google Scholar]
  40. Privalov P. L., Griko YuV, Venyaminov SYu, Kutyshenko V. P. Cold denaturation of myoglobin. J Mol Biol. 1986 Aug 5;190(3):487–498. doi: 10.1016/0022-2836(86)90017-3. [DOI] [PubMed] [Google Scholar]
  41. Rahmelow K., Hübner W., Ackermann T. Infrared absorbances of protein side chains. Anal Biochem. 1998 Mar 1;257(1):1–11. doi: 10.1006/abio.1997.2502. [DOI] [PubMed] [Google Scholar]
  42. Richardson J. M., 3rd, Lemaire S. D., Jacquot J. P., Makhatadze G. I. Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies. Biochemistry. 2000 Sep 12;39(36):11154–11162. doi: 10.1021/bi000610b. [DOI] [PubMed] [Google Scholar]
  43. San Biagio P. L., Bulone D., Emanuele A., Palma M. U. Self-assembly of biopolymeric structures below the threshold of random cross-link percolation. Biophys J. 1996 Jan;70(1):494–499. doi: 10.1016/S0006-3495(96)79595-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. San Biagio P. L., Martorana V., Emanuele A., Vaiana S. M., Manno M., Bulone D., Palma-Vittorelli M. B., Palma M. U. Interacting processes in protein coagulation. Proteins. 1999 Oct 1;37(1):116–120. doi: 10.1002/(sici)1097-0134(19991001)37:1<116::aid-prot11>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]
  45. Smeller L., Rubens P., Heremans K. Pressure effect on the temperature-induced unfolding and tendency to aggregate of myoglobin. Biochemistry. 1999 Mar 23;38(12):3816–3820. doi: 10.1021/bi981693n. [DOI] [PubMed] [Google Scholar]
  46. Tsuda S., Miura A., Gagné S. M., Spyracopoulos L., Sykes B. D. Low-temperature-induced structural changes in the Apo regulatory domain of skeletal muscle troponin C. Biochemistry. 1999 May 4;38(18):5693–5700. doi: 10.1021/bi982936e. [DOI] [PubMed] [Google Scholar]
  47. Vidugiris G. J., Royer C. A. Determination of the volume changes for pressure-induced transitions of apomyoglobin between the native, molten globule, and unfolded states. Biophys J. 1998 Jul;75(1):463–470. doi: 10.1016/S0006-3495(98)77534-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wetzel R. Mutations and off-pathway aggregation of proteins. Trends Biotechnol. 1994 May;12(5):193–198. doi: 10.1016/0167-7799(94)90082-5. [DOI] [PubMed] [Google Scholar]
  49. Wroblowski B., Díaz J. F., Heremans K., Engelborghs Y. Molecular mechanisms of pressure induced conformational changes in BPTI. Proteins. 1996 Aug;25(4):446–455. doi: 10.1002/prot.5. [DOI] [PubMed] [Google Scholar]
  50. Zhang J., Peng X., Jonas A., Jonas J. NMR study of the cold, heat, and pressure unfolding of ribonuclease A. Biochemistry. 1995 Jul 11;34(27):8631–8641. doi: 10.1021/bi00027a012. [DOI] [PubMed] [Google Scholar]
  51. Zipp A., Kauzmann W. Pressure denaturation of metmyoglobin. Biochemistry. 1973 Oct 9;12(21):4217–4228. doi: 10.1021/bi00745a028. [DOI] [PubMed] [Google Scholar]

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